Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease genetically linked to human chromosomal locus 4q35-qter. The genetic abnormality in this region associated with FSHD involves deletions of 3.3 kb repeating sequences (D4Z4 repeats) at the telomere of chromosome 4. However, deletions of these repeats are not sufficient to cause FSHD. Our current understanding is that all symptomatic FSHD patients reportedly have fewer than 10 D4Z4 repeating units and also have inherited the 4qA161 haplotype of 4qA on chromosome 4. Up-regulation of genes either within or in cis to the repeat region and factors associated with the proximity of 4q35 to the nuclear envelope have been proposed as mechanisms, with some experimental support, but gene array and quantitative RT-PCR studies have failed confirm this hypothesis. Thus, the changes in gene expression presumably caused by these deletions remain unclear. I have adopted a combination of proteomic and cell biological approaches to study FSHD muscle. My preliminary results indicate that the soluble fraction of deltoid muscles shows a single protein spot that is present in high amounts in all FSHD samples I have studied, but absent in healthy and diseased controls. The isoelectric point of 5.08, molecular mass of ~34 kDa, and 3 peptide sequences identified this spot as mu-crystallin, also known as NADPH-dependent thyroid binding protein. Immunoblots with mono-specific antibodies to mu-crystallin confirmed its presence at high levels in FSHD deltoid muscles compared to controls. The fact that mu- crystallin is highly over-expressed is consistent with FSHD's autosomal dominant character, which is normally associated with a "gain of function" mutation. As thyroid hormone (T3) plays a central role in the differentiation of muscle, the over-expression of mu-crystallin could contribute to pathogenesis in FSHD in part by altering T3 activity. Remarkably, the inner ear and the retina are both affected in FSHD patients, and mutations in mu- crystallin have been linked to deafness and to retinal defects. Here I propose 3 specific aims to test critically the novel hypothesis that up-regulation of mu-crystallin is specifically linked to, and pathogenic for, FSHD: (i) to examine biopsies from different muscles from age- and sex-matched individuals, and different protein fractions isolated from these samples, to learn if mu-crystallin is consistently present at higher levels in FSHD muscle compared to controls;(ii) to use quantitative RT-PCR and sequence analysis to learn whether mu- crystallin is transcriptionally upregulated in muscles from patients with FSHD and whether there are sequence variations in mu-crystallin transcripts consistently present in FSHD patients, compared to healthy and diseased control muscles. (iii) to use in vivo electroporation and transgenic mice over-expressing varying levels of mu- crystallin to assess the pathological consequences of its over-expression in muscle in vivo. As my new hypothesis challenges existing paradigms, my experiments are high risk in nature. If successful, they will have a significant impact on our understanding of the molecular mechanisms underlying FSHD. PUBLIC HEALTH RELEVANCE: The experiments in this proposal are designed to test the novel hypothesis that an increase in the expression of mu-crystallin (CRYM) is sufficient to cause pathology in skeletal muscle. The impetus for this work stems from my preliminary finding that mu-crystallin is present at abnormally high levels in muscles from patients with the third most common form of muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), as compared to healthy human muscle or muscle from patients with inflammatory myopathies and other muscular dystrophies. My experiments will confirm that the increased expression of mu-crystallin is specific to muscles from FSHD patients, whether its up-regulation occurs at the transcriptional level, and whether chronic over-expression of this protein can cause muscular dystrophy in otherwise normal mice. As my new hypothesis challenges existing paradigms, my experiments are high risk in nature. If successful, they will have a significant impact on our understanding of the molecular mechanisms underlying FSHD.